Hostname: page-component-cd9895bd7-7cvxr Total loading time: 0 Render date: 2024-12-21T15:52:26.267Z Has data issue: false hasContentIssue false
  • English
  • Français

Cloning and eukaryotic expression of olfaction-related Gqα-protein gene in English grain aphid (Sitobion avenae)

Published online by Cambridge University Press:  30 October 2009

Fan Jia ,
Chen Ju-Lian ,
Cheng Deng-Fa  and
Sun Jing-Rui
Fan Jia
Affiliation:
State Key Laboratory for Biology of Plant Diseases and Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
Chen Ju-Lian*
Affiliation:
State Key Laboratory for Biology of Plant Diseases and Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
Cheng Deng-Fa
Affiliation:
State Key Laboratory for Biology of Plant Diseases and Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
Sun Jing-Rui
Affiliation:
State Key Laboratory for Biology of Plant Diseases and Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
*
*Corresponding author. E-mail: jlchen@ippcaas.cn or cjl2000@hotmail.com
Get access Rights & Permissions [Opens in a new window]

Abstract

Based on conserved homologous amino-acid sequences of the Gq protein α subunit in arthropods, a pair of degenerate primers were designed to amplify the gene from the English grain aphid (Sitobion avenae), using reverse transcriptase polymerase chain reaction (RT-PCR) and (3′/5′)-rapid amplification of cDNA ends (3′/5′ RACE) techniques. A Gqα protein was obtained from the alate adult aphids. The open reading-frame was 1062 bp, encoding 352 amino-acid residues with a calculated molecular weight of 40.8 kDa. The cDNA sequence was deposited in GenBank with accession no. EF638906. The deduced amino-acid sequence of Gqα shared a high identity (≥82.17%) with reported Gqα from other insects and even vertebrates, and had the typical characteristics of Gqα protein. In order to explore the function of the Gqα gene, a eukaryotic expressional system (baculovirus expression vector system, BEVS) was constructed by TOPO and Gateway techniques. After the recombinant reaction occurring between pUC-Gqα and the Gateway-adapted baculovirus DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV), the construct recombinant viruses containing V5-His6Gqα were transfected singly into the insect cell line of Tn-5B1-4. After collecting the infected cell, detection was conducted by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The result showed that the system comprising recombinant baculovirus and Tn could express Gqα protein successfully.

Keywords

English grain aphidprotein related with olfactoryGqα proteinGqα gene cloningbaculovirus expression vector system (BEVS)
Type
Research Papers
Information
Chinese Journal of Agricultural Biotechnology , Volume 6 , Issue 2 , August 2009 , pp. 111 - 117
Copyright
Copyright © China Agricultural University 2009

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Chen, JL, Weng, GZ and Ni, HX (2001) The advancement of G protein and coupled signal transduction pathways. Chinese Journal of Biotechnology 17(2): 113–117 (in Chinese with English abstract).Google ScholarPubMed
Chen, JL, Ni, HX, Sun, JR and Weng, GZ (2003) G protein β1γ2 subunit purification and their interaction with adenylyl cyclase. Sciences in China 46(2): 212–223 (in Chinese).Google ScholarPubMed
Clyne, PJ, Warr, CG and Freeman, MR (1999) A novel family of divergent seven-transmembrane proteins: candidate odorant receptors in Drosophila. Neuron 22: 327–338.CrossRefGoogle ScholarPubMed
Fadool, DA, Estey, SJ and Ache, BW (1995) Evidence that Gq-protein mediates excitatory odor transduction in lobster olfactory receptor neurons. Chemical Senses 20: 489–498.CrossRefGoogle ScholarPubMed
Gao, Q and Chess, A (1999) Identification of candidate Drosophila olfactory receptors from genomic DNA sequence. Genomics 60: 31–39.CrossRefGoogle ScholarPubMed
Hill, SJ (2006) G-protein-coupled receptors: past, present and future. British Journal of Pharmacology 147: S27–S37.CrossRefGoogle ScholarPubMed
Holt, RA, Subramanian, GM, Halpern, A, et al. (2002) The genome sequence of the malaria mosquito Anopheles gambiae. Science 298(5591): 129–149.CrossRefGoogle ScholarPubMed
Hubbard, KB and Hepler, JR (2006) Cell signaling diversity of the Gqα family of heterotrimeric G proteins. Cell Signal 18(2): 135–150.CrossRefGoogle ScholarPubMed
Jacquin-Joly, E, François, MC, Burnet, M, Lucas, P, Bourrat, F and Maida, R (2002) Expression pattern in the antennae of a newly isolated lepidopteran Gq protein α subunit cDNA. FEBS Letter 269: 2133–2142.Google ScholarPubMed
Jiang, HL, Chen, JL, Ni, HX and Cheng, DF (2006) Cloning of gene encoding G-protein gamma subunit and tissue distribution in the English grain aphid, Sitobion avenae. Proceedings of the 2006 Annual Meeting of China Association for Science and Technology: 53–57 (in Chinese with English abstract).Google Scholar
Jiang, HL, Chen, JL, Ni, HX and Cheng, DF (2005) Cloning of gene encoding G-protein beta subunit and tissue distribution in the English grain aphid, Sitobion avenae. Acta Phytophylacica Sinica (Journal of Plant Protection) 32(1): 1–6 (in Chinese with English abstract).Google Scholar
Lambright, DG, Sondek, J, Bohm, A, Skiba, NP, Hamm, HE and Sigler, PB (1996) The 2.0 Å crystal structure of a heterotrimeric G protein. Nature 379(6563): 311–319.CrossRefGoogle ScholarPubMed
Li, ZX (2004) Cloning identification and expression profiling of the cDNAs of odorant-binding proteins in the malaria mosquito, Anopheles gambiae. Acta Entomologica Sinica 47(4): 417–423 (in Chinese with English abstract).Google Scholar
Munger, SD, Gleeson, RA, Aldrich, HC, Rust, NC, Aches, BW and Greenberg, M (2000) Characterization of a phosphoinositide-mediated odor transduction pathway reveals plasma membrane localization of an inositol 1,4,5-triphosphate receptor in lobster olfactory receptor neurons. Journal of Biological Chemistry 27: 20450–20457.CrossRefGoogle Scholar
Nault, LR, Edwards, LJ and Styer, WE (1973) Aphid alarm pheromone secretion and reception. Environmental Entomology 2: 101–105.CrossRefGoogle Scholar
Slessareva, JE, Routt, SM, Temple, B, Bankaitis, VA and Dohlman, HG (2006) Activation of the phosphatidylinositol 3-kinase Vps34 by a G protein alpha subunit at the endosome. Cell 126(1): 191–203.CrossRefGoogle Scholar
Talluri, S, Bhatt, A and Smith, DP (1995) Identification of a Drosophila G protein alpha subunit (dGq alpha-3) expressed in chemosensory cells and central neurons. Proceedings of the National Academy of Sciences of the USA 92: 11475–11479.CrossRefGoogle ScholarPubMed
Vosshall, LB, Amrein, H, Morozov, PS, Rzhetsky, A and Axel, R (1999) A spatial map of olfactory receptor expression in the Drosophila antennae. Cell 96: 725–736.CrossRefGoogle Scholar
Wallet, MA, Coleman, DE, Lee, E et al. (1995) The structure of the G protein heterotrimer Gi alpha 1 β 1 γ2. Cell 83: 1047–1058.CrossRefGoogle Scholar
Wang, GR, Wu, KM and Guo, YY (2003) Cloning expression and immunocytochemical localization of a general odorant-binding protein from Helicoverpa armigera. Insect Biochemistry and Molecular Biology 33: 115–124.CrossRefGoogle ScholarPubMed